For decades, tree breeders have faced the dilemma of how to mate a select group of individuals (parents) for the next generation of genetic improvement (see step 3 of Table 1). Initially, plant breeders have no problem making selections of individual trees that have desired phenotypic traits from progeny tests or commercial tree stands. The difficulty faced by tree breeders is how to design a plant breeding program using the select individuals to accomplish tree improvement through future generations. The chosen mating design is usually a compromise as no single plant breeding design has been found that best meets all of the objectives of a long-term tree breeding program (Namkoong, U.S.D.A. Forest Service Technical Bulletin No. 1588:342 (1979)).
TABLE 1Process flow for a typical genetic improvement program for forest trees.
In general, plant breeding designs share the following common objectives:                1. Accurately estimate the breeding value of the parents being bred for purposes of recommending which to include in a production seed orchard or for purposes of determining which crosses to make new selections in for the next generation of breeding, i.e., those crosses between high breeding value parents. This selection of parents based on progeny performance is also called “backward” selection. See step 9, Table 1.        2. Produce a set of crosses that will allow high genetic gain potential from selection of the best individuals within the best crosses for the next generation of breeding, often called “forward” selection. See step 8, Table 1.        3. Allow full pedigree control where both male and female parents are known for new selections in order to control the level of inbreeding.        4. Generate sound estimates of genetic parameters in field trials such as heritability, general and specific combining ability, genetic correlations among traits and genotype-environment interaction.        5. Do all of the above efficiently in order to keep breeding and testing costs at an acceptable level.        
The most obvious breeding scheme to achieve all of the above objectives would be to cross every parent with every other parent (full diallel cross) but that alternative is almost always rejected in operational breeding improvement programs due to the fact that it is impractical to make and test so many crosses. The number of total selections may be on the order of a few hundred trees and the number of possible crosses is in the thousands, a number that may be prohibitively expensive to breed and test. The latter is especially true for tree species which, due to their large size, require special equipment to do breeding and a large area for testing individual plants. Consequently, forest geneticists have sought other mating and testing designs that achieve their primary objectives as efficiently as possible.
Mating designs that have been commonly compared in the literature are open pollination versus controlled pollination and, for the latter, full-sib crossing versus polymix (a mixture of pollen from several males) crossing (Bridgwater, In: Fins et al. (eds.) Handbook of Quantitative Forest Genetics, Kluwer Academic, Dordrect, The Netherlands, pp. 140-194 (1992)). The most common method of quantitative comparisons of these mating designs in the literature is to hold the number of parents bred and number of progeny tested from crosses among them as constants in each generation of breeding and then determine how well each design estimates genetic parameters and their genetic gain potential. A synopsis of these comparisons of breeding methods follows.
Open Pollination
Open pollination (OP) in forestry is usually assumed to take place in a progeny test where only a hand full of new selections are found among several hundred or a few thousand other individuals. OP breeding gives good estimation of parental breeding values and heritability estimates (Bridgwater, In: Fins et al. (eds.) Handbook of Quantitative Forest Genetics, Kluwer Academic Pub., Dordrect, The Netherlands. pp. 140-194 (1992); Cotterill, Proc. IUFRO Conference on Breeding Theory, Progeny Testing and Seed Orchards, Williamsburg, Va., USA., pp. 144-149 (1986); Cotterill, Silv. Genet. 35(5-6):212-223 (1986b); White, In: Proc QFRI-IUFRO Conf, Tree Improvement for Sustainable Tropical Forestry, Caloundra, Australia, pp. 110-117 (1996)).
The genetic gain potential from using the OP families for the next generation of progeny testing is weak since the select parents are mated with a nonselect population from the previous generation (Cotterill, Proc. IUFRO Conference on Breeding Theory, Progeny Testing and Seed Orchards, Williamsburg, Va., USA., pp. 144-149 (1986a); Cotterill, Silv. Genet. 35(5-6):212-223 (1986); van Buijtenen et al., In: Proc IUFRO Joint Meeting of Genetic Working Parties on Advanced Generation Breeding, Bordeaux, France, pp. 11-29 (1976)). This and other disadvantages such as lack of full pedigree control and the inability to estimate specific combining ability have resulted in limited use of OP breeding in forestry except in programs where limited resources dictate a simple and low cost approach.
Full-Sib Crossing
There are many mating patterns that have been discussed and analyzed in the literature including full-diallel, cousin mating, single-pair, nested, factorial, disconnected half-diallels and circular systems (Bridgwater, In: Fins et al. (eds.). Handbook of Quantitative Forest Genetics, Kluwer Academic Pub., Dordrect, The Netherlands. pp. 140-194 (1992); Burdon and van Buijtenen, Can. J. For. Res. 20:1664-1671 (1990); Cotterill, Proc. IUFRO Conference on Breeding Theory, Progeny Testing and Seed Orchards, Williamsburg, Va., USA., pp. 144-149 (1986); Cotterill, Silv. Genet. 35(5-6):212-223 (1986); Huber et al., For. Sci. 38(4):757-776 (1992); Pederson, Theor. Appl. Genet. 42:371-377 (1972); van Buijtenen and Burdon, Can. J. For. Res. 20:1648-1663 (1990)). Of these, the last three have been commonly used in forest tree improvement because they provide several strengths including reasonably good parameter estimation (general and specific combining ability, heritability, breeding value of parents), provide a foundation for reasonably good genetic gain and provide full pedigree control. In these designs each parent is usually mated to four to six of the other parents in the breeding group.
The weaknesses of the full-sib systems are the amount of work involved in breeding and testing and the fact that they are not the best designs for estimating breeding value nor do they provide the best foundation for selection for the next generation of breeding. These limitations stem from the relatively few crosses for each parent. For a given population size for the progeny test of the crosses it is generally better to have many crosses per parent for precise parameter estimation and high genetic gain potential from forward selection (Pederson, Theor. Appl. Genet. 42:371-377 (1972); White, In: Proc QFRI-IUFRO Conf, Tree Improvement for Sustainable Tropical Forestry, Caloundra, Australia, pp. 110-117 (1996)) especially when selection emphasis is heavily on family versus individual within family under low heritability situations. Performance of a small number of crosses can result in inaccurate breeding value estimation, especially if there is significant specific combining ability (SCA) in the breeding population (Burdon and van Buijtenen, Can. J. For. Res. 20:1664-1671 (1990); van Buijtenen and Burdon, Can. J. For. Res. 20:1648-1663 (1990)). Genetic gain for the next generation is limited due to the fact that the best parents, among those being bred, may not have been frequently mated to the other best parents due to chance. Nonetheless, few crosses and many individuals per cross are usually opted due to the high cost of making and testing the crosses. Although full pedigree is known, the limited number of crosses usually means that there is a tendency to select from within a few good crosses. However, there are limitations in so doing because there can often be common parentage among those crosses such that inbreeding concerns force selection from mediocre families, thus limiting genetic gain.
Polymix Crossing
Polymix crossing is done by mixing pollen from several males and applying the pollen to isolated females. One of the advantages to polymix crossing is the simplicity of crossing and subsequent testing of relatively few crosses. Polymix crossing is sometimes considered for parental breeding value (BV) estimation only (Burdon and van Buijtenen, Can. J. For. Res. 20:1664-1671 (1990); White, In: Proc QFRI-IUFRO Conf, Tree Improvement for Sustainable Tropical Forestry, Caloundra, Australia, pp. 110-117 (1996)), in which case the source of pollen may or may not be made up of the parents in the breeding group, or as a complete breeding system for BV estimation and as a foundation for selection of individuals for advanced generation breeding. In the latter case it is preferable to use pollen from the select group in order to keep genetic gain potential high (Cotterill, Proc. IUFRO Conference on Breeding Theory, Progeny Testing and Seed Orchards, Williamsburg, Va., USA., pp. 144-149 (1986); Cotterill, Silv. Genet. 35(5-6):212-223 (1986); Burdon and Shelbourne, NZ J. For. Sci. 1(2):174-193 (1971); Kerr, Theor. Appl. Genet. 96:484-493 (1998); Shelbourne, Tech Pap. No. 55, New Zealand Forest Service, 44 pp. (1969)). PMX crossing provides excellent estimation of breeding value and general combining ability (GCA), but not usually specific combining ability (SCA) (Bridgwater, In: Fins et al. (eds.). Handbook of Quantitative Forest Genetics, Kluwer Academic Pub., Dordrect, The Netherlands. pp. 140-194 (1992); Huber et al., For. Sci. 38(4):757-776 (1992)). However, Janssens (Silv. Gen. 29(3-4):138-140 (1980)) devised a nested PMX design that allows for estimation of SCA variance at the population level but obviously does not estimate SCA effects for a specific cross. Genetic gain potential for forward selection is good but not as high as that offered by the commonly used full-sib systems mentioned above. The gain potential from PMX crossing is 70% to over 90% of the potential for full-sib systems (Cotterill, Proc. IUFRO Conference on Breeding Theory, Progeny Testing and Seed Orchards, Williamsburg, Va., USA., pp. 144-149 (1986); Cotterill, Silv. Genet. 35(5-6):212-223 (1986); Kerr, Theor. Appl. Genet. 96:484-493 (1998); Shelbourne, Tech Pap. No. 55, New Zealand Forest Service, 44 pp. (1969); van Buijtenen and Burdon, Can. J. For. Res. 20:1648-1663 (1990)), depending on the magnitude of genetic parameters, with increasing heritability and SCA favoring PMX crossing (van Buijtenen and Burdon, Can. J. For. Res. 20:1648-1663 (1990)).
The primary reason that PMX systems result in less genetic gain from advanced generation selections versus full-sib systems is due to the fact that the paternal parent's GCA is unknown. When individual selections are made within the best full-sib crosses for the next generation of breeding they are commonly chosen on the basis of an index that includes the following information:Individual Breeding Value=female GCA+male GCA+h2w(individual deviation within the full-sib cross)  (1)
Where: h2w=within cross heritability.
For the PMX cross individual value is estimated as follows:Individual Breeding Value=female GCA+h2w(individual deviation within the PMX cross)  (2)
Selection within PMX crosses does not have the benefit of knowing the male GCA, but the heritability for and variation within a PMX crossing scheme is greater than that for full-sib crosses due to the fact that a PMX cross is actually made up of the variation among many full-sib crosses as well as the variation within those crosses. In balance, lack of information on male GCA results in the somewhat reduced gain efficiency of PMX breeding and testing systems mentioned above. Lack of full pedigree control and the possibility that a few males may be represented among selections for the next generation breeding resulting in possible inbreeding depression is another drawback that has limited use of PMX crossing as a stand alone system (Bridgwater, In: Fins et al. (eds.). Handbook of Quantitative Forest Genetics, Kluwer Academic Pub., Dordrect, The Netherlands. pp. 140-194 (1992)).
Combined Systems
Many plant breeders have gone to a combination of mating designs in order to take advantages of the strengths of each and to offset, to some degree, the limitations of any one design (van Buijtenen and Burdon, Can. J. For. Res. 20:1648-1663 (1990)). The major tree improvement cooperatives for pine species in the southeastern U.S. have gone to a complementary system consisting of PMX crossing for parental breeding values and full-sib crossing from which selections are made for the next generation of breeding (Lowe and van Buijtenen, In: Proc IUFRO Conf on Breeding and Testing Theory, Progeny Testing and Seed Orchards, Williamsburg, Va., pp. 98-106 (1986); McKeand and Bridgwater, In: Proc IUFRO Resolving Tropical Forest Resource Concerns Through Tree Improvement, Gene Conservation, and Domestication of New Species, Cali, Colombia, pp. 234-240 (1992); White et al., Silv. Genet. 42:359-371 (1993)). Full-sib crossing is typically a circular or disconnected half diallels design. While offering many advantages, these systems are costly of time and resources. Furthermore, they do not overcome the limitation of few crosses per parent and the fact that the best parents may not often be mated with the other best parents, thus limiting gain from selection with crosses and/or creating relatedness limitations among advanced generation selections.
In summary, the type of plant breeding strategy selected for use in a plant breeding program depends upon a number of different factors. However, a common goal of a plant breeding program is to generally use a breeding method that will result in the largest possible genetic gain using the smallest number of breeding crosses and the least amount of time and money. What is needed in the art of plant breeding is a breeding method that will reduce the number of generations that are required in a plant breeding program to achieve an improved plant variety yet at the same time be cost and time effective.
The present invention provides a new plant breeding method that utilizes cost and time effective parental pedigree determination using molecular analysis in conjunction with phenotypic scores to efficiently select elite progeny plants for use in the next generation of plant breeding. The molecular pedigree methods used in the present invention overcome the heretofore primary limitations of PMX breeding and testing which are lack of male pedigree control and lack of male GCA information for advanced generation selection. More specifically, the present invention provides a plant breeding design which utilizes PMX crosses in conjunction with molecular marker technology to determine pedigree. Given a sufficient number of reliable, polymorphic molecular markers, and modest care in creation of pollen polymixes, paternity of all PMX progeny can be unambiguously determined through molecular analysis. Therefore, the present invention provides a plant breeding method that allows for pedigree control and estimation of breeding values of the parental plants and their progeny.